Abstract

Aim

Glioblastoma multiforme (GBM) are hard to treat cancers with minimal survival rate. Standard treatments are not efficient, making novel strategies a necessity. One of the antigens overexpressed on GBM cells is c-Met, which we intend to target immunotherapeutically using CARs. CAR is a chimeric conglomeration of single chain fragment variable (scFv) of an antibody, with moieties of T cell receptor which is capable of generating effector functions in a T cell when engrafted. Here, we want to generate a CAR targeting c-met, select the hinge region allowing for better antigen recognition and co-stimulatory molecules allowing for better CAR efficacy. In addition, we want to be able to selectively ablate CAR T cells in advent of adverse reactions by inserting a suicide gene in the CAR backbone.

Methods

Several GBM cell lines were tested for c-Met expression and U251 was selected as a model cell line. HB11895™ hybridoma was used to amplify variable regions by PCR, using degenerate primers. A (G4S)3 liker was introduced between these variable regions to generate a scFv. CAR backbone was designed and was optimized for different hinge lengths and different co-stimulatory molecules. A suicide gene (icaspase 9) was also incorporated in the backbone. CAR molecules were integrated in a lentivector to generate virus, to transduce T cells to express c-Met specific CARs. Specificity of CAR T cells was determined by detecting IFN-ɣ, TNF-α and IL2 by intracellular cytokine staining (ICS) post c-Met exposure.

Results

scFv was extracted from c-Met hybridoma. CAR backbone was designed with appropriate restriction sites allowing 4 hinge and 3 co-stimulatory domain variants. Lentiviral production and purification was performed and T cells were transduced at ≥90 % efficiency. A specific release of IFN-ɣ, TNF-α and IL2 was noted in ICS. This led to the selection of optimized hinge. More than 95% CAR T cells were ablated by suicide gene triggering.

Conclusions

We succeeded in generating c-Met CAR T cells which were specifically activated upon c-Met binding. We are now going to analyze the efficiency of our CAR T cells in vivo on a glioma mouse model for pre-clinical testing, which in turn paves way to clinical preface.

Clinical trial identification

Disclosure

P.-Y. Dietrich: Immatics's research grant. All other authors have declared no conflicts of interest.